An efficient method for cloning human autoantigen-specific T cells

T-cell clones are valuable tools for investigating T-cell specificity in infectious, autoimmune and malignant diseases. T cells specific for clinically-relevant autoantigens are difficult to clone using traditional methods. Here we describe an efficient method for cloning human autoantigen-specific CD4+ T cells pre-labelled with CFSE. Proliferating, antigen-responsive CD4+ cells were identified flow cytometrically by their reduction in CFSE staining and single cells were sorted into separate wells. The conditions (cytokines, mitogens and tissue culture plates) for raising T-cell clones were optimised. Media supplemented with IL-2+IL-4 supported growth of the largest number of antigen-specific clones. Three mitogens, PHA, anti-CD3 and anti-CD3+anti-CD28, each stimulated the growth of similar numbers of antigen-specific clones. Cloning efficiency was similar in flat- and round-bottom plates. Based on these findings, IL-2+IL-4, anti-CD3 and round-bottom plates were used to clone FACS-sorted autoantigen-specific CFSE-labelled CD4+ T cells. Sixty proinsulin- and 47 glutamic acid decarboxylase-specific clones were obtained from six and two donors, respectively. In conclusion, the CFSE-based method is ideal for cloning rare, autoantigen-specific, human CD4+ T cells.     

Huntington disease-linked locusD4S111 exposed as the α-l-iduronidase gene

-l-Iduronidase (IDUA) has been intensively studied due to its causative role in mucopolysaccharidosis type I (Hurler, Scheie and Hurler/Scheie syndromes). The recent cloning of a human IDUA cDNA has resulted in a reevaluation of the chromosomal location of this gene. Previously assigned to chromosome 22, IDUA now has been localized to 4p16.3, the region of chromosome 4 associated with Huntington's disease (HD). The existence of a battery of cloned DNA, physical map information, and genetic polymorphism data for this region has allowed the rapid fine mapping of IDUA within the terminal cytogenetic band of 4p. IDUA was found to be coincident with D4S111, an anonymous locus displaying a highly informative multiallele DNA polymorphism. This map location, 1.1×10⁶ bp from the telomere, makes IDUA the most distal cloned gene assigned to 4p. However, it falls within a segment of 4p16.3 that has been eliminated from the HD candidate region, excluding a role for IDUA in this disorder.     

Tetracycline-regulated gene expression following direct gene transfer into mouse skeletal muscle

For most experimental and therapeutic applications of gene transfer, regulation of the timing and level of gene expression is preferable to constitutive gene expression. Among the systems that have been developed for pharmacologically controlled gene expression in mammalian cells, the bacterial tetracycline (tet)-responsive system has the advantage that it is dependent on a drug (tet) that is both highly specific and non-toxic. The tet-responsive system has been previously used to modulate expression of cell cycle regulatory proteins in cultured cells, reporter genes in plants and transgenic mice and reporter genes directly injected into the heart. Here we show that orally or parenterally administered tet regulates expression of tet-responsive plasmids injected directly into mouse skeletal muscle. Reporter gene expression was suppressed by two orders of magnitude in the presence of tet, and that suppression was reversed when tet was withdrawn. These data show that skeletal muscle offers an accessible and well characterized target tissue for tet-controlled expression of genesin vivo, suggesting applications to developmental studies and gene therapy.     

Impaired Microvascular Function in Normal Children: Effects of Adiposity and Poor Glucose Handling

Clustering of cardiovascular risk factors is thought to occur early in life. The endothelium is an important regulator of microvascular function. We investigated the relationship between microvascular function and cardiovascular risk factors in 145 normal, healthy children aged 11-14 years. Skin microvascular responses, measured using laser Doppler imaging, to iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP), were negatively correlated with percentage body fat ( r =−0.20, P < 0.05 and r =−0.18, P < 0.05, respectively). Subjects were stratified into quintiles based on 2-h, post-feeding glucose levels. Subjects in the upper glucose quintile (range 7.4-11.4 mmol l⁻¹) showed significantly lower vasodilatation to both ACh (   P < 0.005  ) and SNP (   P < 0.02  ) than those in the lower quintile (range 3.9-4.9 mmol l⁻¹). Waist-to-hip ratio and the fasting insulin resistance index were significantly greater in subjects in the upper quintile than those in the lower quintile ( P < 0.001 and P < 0.05, respectively). Additionally, in subjects in the upper glucose quintile, fasting triglyceride correlated with fasting insulin ( r = 0.59, P < 0.001) and with the fasting insulin resistance index ( r = 0.49, P < 0.009), and plasma levels of cholesterol and 2-h glucose were also correlated ( r = 0.40, P < 0.05). In a cross-section of normal children, microvascular function was negatively associated with adiposity. Additionally, in a subgroup of subjects, there was a clustering of high post-feeding glucose, impaired microvascular function, increased insulin resistance and higher central fat distribution. These findings suggest that risk factors for adult cardiovascular disease begin to cluster in normal children, which might have important consequences for development of atherosclerosis later in life.     

Susceptibility of anaerobic bacteria to antimicrobial agents

The antimicrobial susceptibility of 1,117 clinical isolates of anaerobic bacteria was determined by the agar dilution technique. Metronidazole was the most active agent; only Propionibacterium acnes and Actinomyces sp. isolates were resistant. Clindamycin and chloramphenical were the next most effective agents. Beta-lactam antibiotics, with the exception of penicillin, were active against most anaerobes other than the Bacteroides fragilis group. At a breakpoint of 8 mg/l, 25% of Fusobacterium spp. and 30% of the non-fragilis Bacteroides spp. were resistant to penicillin. The highest resistance to beta-lactams was seen in the B. fragilis group. Within the indole-positive members of the group, resistance rates of 71% were seen for cefoxitin, 49% for moxalactam, 79% for cefotaxime, 22% for piperacillin and 89% for penicillin. We conclude that metronidazole has the most predictable in vitro activity against common clinical anaerobic isolates and that resistance to beta-lactams was frequent and of potential clinical importance as these latter agents are frequently used in the prophylaxis and therapy of mixed anaerobic infections.     

Comparison of two laboratory tests in the control of anticoagulant therapy

A prospective, randomized trial is described in which the usefulness of two tests in the control of anticoagulant therapy is compared. Fifty-two patients were controlled by the one-stage prothrombin time and 55 by the activated partial thromboplastin time. There was no significant difference in the incidence of bleeding between the two groups. When bleeding did occur, it was more often reflected by prolongation of the prothrombin time than of the activated partial thromboplastin time. The prothrombin time was found to have some practical advantages over the activated partial thromboplastin time.     

Implementation,generalization and long-term results of the "choosing well" diabetes self-management intervention

Prerequisites for translating intervention research findings into practice are maintenance of results, generalization of effects and consistency of implementation. This report presents 12 months follow-up information on a randomized 2x2 factorial trial evaluating the incremental effects of adding (1) telephone follow-up or (2) a community resources utilization component to a basic touchscreen computer-assisted dietary goal-setting intervention for 320 type 2 diabetes patients. All conditions evidenced significant improvement from baseline to the 12 months follow-up across behavioral, biological and psychosocial measures. There were few consistent differences between conditions, but results were robust across interventionists and clinics. The telephone follow-up component appeared to enhance long-term results on some measures. When considered along with earlier results from a randomized trial that included a control condition without goal setting, it is concluded that this basic goal-setting intervention can be consistently implemented by a variety of interventionists and produce lasting improvements.     

Analysis of the operation of the SCD Response intermittent compression system

The work assessed the performance of the Kendall SCD Response intermittent pneumatic compression system for deep vein thrombosis prophylaxis, which claimed to set its cycle according to the blood flow characteristics of individual patient limbs. A series of tests measured the system response in various situations, including application to the limbs of healthy volunteers, and to false limbs. Practical experimentation and theoretical analysis were used to investigate influences on the system functioning other than blood flow. The system tested did not seem to perform as claimed, being unable to distinguish between real and fake limbs. The intervals between compressions were set to times unrealistic for venous refill, with temperature changes in the cuff the greatest influence on performance. Combining the functions of compression and the measurement of the effects of compression in the same air bladder makes temperature artefacts unavoidable and can cause significant errors in the inter-compression interval.     

Chagas'' disease.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of morbidity in many countries in Latin America. The important modes of transmission are by the bite of the reduviid bug and blood transfusion. The organism exists in three morphological forms: trypomastigotes, amastigotes, and epimastigotes. The mechanism of transformation and differentiation is currently being explored, and signal transduction pathways of the parasites may be involved in this process. Parasite adherence to and invasion of host cells is a complex process involving complement, phospholipase, penetrin, neuraminidase, and hemolysin. Two clinical forms of the disease are recognized, acute and chronic. During the acute stage pathological damage is related to the presence of the parasite, whereas in the chronic stage few parasites are found. In recent years the roles of tumor necrosis factor, gamma interferon, and the interleukins in the pathogenesis of this infection have been reported. The common manifestations of chronic cardiomyopathy are arrhythmias and thromboembolic events. Autoimmune, neurogenic, and microvascular factors may be important in the pathogenesis of the cardiomyopathy. The gastrointestinal tract is another important target, and "mega syndromes" are common manifestations. The diagnosis and treatment of this infection are active areas of investigation. New serological and molecular biological techniques have improved the diagnosis of chronic infection. Exacerbations of T. cruzi infection have been reported for patients receiving immuno-suppressive therapy and for those with AIDS.     

Passive protection to bovine rotavirus (BRV) infection induced by a BRV VP8* produced in plants using a TMV-based vector

Summary. We have previously reported on the use of a tobacco mosaic virus (TMV) vector TMV-30B to express foreign viral antigens for use as experimental immunogens. Here we describe the development of an improved TMV-30B vector that adds a sequence of 7 histidine residues to the C-terminus of recombinant proteins expressed in the vector. We used this TMV-30B-HISc vector to express the VP8* fragment of the VP4 protein from bovine rotavirus (BRV) strain C-486 in plants. Recombinant VP8* protein was purified from N. benthamiana leaves at 7 days post-inoculation by immobilized metal affinity chromatography. The plant-produced VP8* was initially detected using anti-His tag mAb and its antigenic nature was confirmed using both monoclonal and polyclonal specific antisera directed against BRV. Adult female mice, inoculated by the intraperinoteal route with an immunogen containing 4µg of recombinant VP8*, developed a specific and sustained response to the native VP8* from the homologous BRV. Eighty five percent of suckling mice from immunized dams that were challenged with the homologous virus at the fifth day of age were protected from virus as compared to 35% of the pups from mothers immunized with a control protein. These results demonstrate that the plant-produced VP8* was able to induce passive protection in the new born through the immunization of dams. This suggests that the technology presented here provides a simple method for using plants as an inexpensive alternative source for production of recombinant anti-rotavirus antigens.Authors contributed equally to the results presented in this report.